1142
Biochemistry
R. A. GOLDSBY AND P. G. HEYTLER
Roland, J. F., and Grove, A. M. (1954), A d . Chem. 26, 502. Spackman, D. H., Stein, W. H., and Moore, S. (1958), A d . Chem. 30,1190.
Spies, J. R., and Chambers, D. C. (1949), Anal. Chem. 21,. 1249. Stein, W. H., and Moore, S. (1949-50), cited by Craig, L. C. in Fortschr. Chem. Forsch. 1,312.
Uncoupling of Oxidative Phosphorylation by Carbonyl Cyanide Phenylhydrazones. 11. Effects of Carbonyl Cyanide rn-Chlorophenylhydrazone on Mitochondrial Respiration R. A. GOLDSBY AND P. G. HEYTLER From the Central Research Department,* E. I . du Pont de Nemours and Company Received March 25, 1963
The effects of carbonyl cyanide m-chlorophenylhydrazne (m-Cl-CCP) on respiration in rat liver mitochondria has been studied polarographically and manometrically. Experiments with the oxygen electrode have shown that 2 HM m-C1-CCP abolishes respiratory control when P-hydroxybutyrate, a glutamate-malate mixture, or succinate is used as a substrate. A similar concentration of m-C1-CCP reverses the inhibition of respiration by oligomycin. The depression by m-C1-CCP of succinate oxidation has been shown to be prevented by addition of either cysteine sulfinic acid or lipoic acid. However, neither of these agents protects against uncoupling by m-C1-CCP. The ability of a fixed amount of m-C1-CCP to abolish respiratory control and lower the P/O ratio is dependent on the amount of mitochondrial protein in the system. However, respiratory control is somewhat more sensitive to m-Cl-CCP than is phosphate esterification. The ATP-Pi3* exchange reaction is also inhibited by m-C1-CCP. The possible site of carbonyl cyanide phenylhydrazone action is discussed in relation to these data. The phenomenon of respiratory control as defined and studied by Chance and his collaborators (Chance and Williams, 1956) has been of great value in the study of oxidative phosphorylation. Through studies of respiratory control, it is possible to discriminate between substances that inhibit oxidative phosphorylation, such as guanidines (Pressman, 1963; Hollunger, 1955), and true uncouplers of this process, i.e., 2,4DNP. Polarographic observation of respiration provides useful information about the mode of action of inhibitory substances known to prevent phosphate esterification; e.g., Estabrook's (1961) studies with oligomycin. With these considerations in mind, a study of the effects of m-C1-CCP on mitochondrial respiration and respiratory control has been conducted and is reported here. In addition to studies of CCP effeds on respiration, it was felt that useful information might be obtained by a study of the ATP-Pi32 exchange reaction described by Boyer et. al. (1956). An earlier paper (Heytler, 1963) reported the effect of m-C1-CCP on several other aspects of mitochondrial oxidative phosphorylation. EXPERIMENTAL Mitochondria used in these studies were obtained from the livers of young adult male rats. The method of isolation is outlined in the previous paper (Heytler, 1963). Oxidation of substrate was measured either by direct determination of oxygen uptake or in some cases by colorimetric measurements of acetoacetate (Walker, 1954). Direct measurements of oxygen uptake were obtained manometrically by standard Warburg technique, or polarographically with a modified Clark electrode (Yellow Springs Instrument Company, Yellow springs, Ohio). Determinations of phosphate uptake were made by measuring the disappearance of inorganic phosphate
* Contribution No. 839.
from the medium (Taussky and Schoor, 1953). Protein determinations were made according to the method of Lowry etal. (1951). The ATP-Pi32exchange was conducted under a nitrogen atmosphere a t 20". Incorporation of labeled phosphate into ATP was measured after removal of inorganic phosphate by solvent extraction (Nielson and Lehninger, 1955). Radioactivity determinations were performed by drying aliquots of P32-containing solutions on aluminum planchets and counting with a thinwindow GM tube. The carbonyl cyanide m-chlorophenylhydramne used in this study was synthesized by Dr. W. W. Prichard of this laboratory by the method described in a previous report (Heytler and Prichard, 1962). The oligomycin was the generous gift of Dr. H. A. Lardy. All other chemicals used in these investigations were obtained from commercial sources in the highest purity routinely available. RESULTS
Polarographic Studies of Coupled Mitmhurulria.The polarographic tracing of Figure 1 shows that the mitochondria used in these studies exhibit respiratory control' with respect to both ADP and Pi. Figure 2 shows that the respiratory control of P-hydroxybutyrate oxidation by ADP is abolished by 2 PM m-Cl-CCP. As shown in Figure 3, the respiration of a coupled mitochondrial system deficient in ADP and Pi is markedly stimulated by 2 PM m-C1-CCP. The stimulation of respiration is seen immediately (
